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Overview The Gram stain procedure was originally developed by the Danish physician Hans Christian Gram to differentiate pneumococci from Klebsiella pneumonia. In brief, the
procedure involves the application of a solution of iodine (potassium
iodide) to cells previously stained with crystal violet or gentian
violet. This procedure produces "purple colored iodine-dye complexes" in the cytoplasm
of bacteria. The cells that
are previously stained with crystal violet and iodine are
next treated with a decolonizing agent such as
95% ethanol or a mixture of acetone
and alcohol. The difference between Gram-positive and Gram-negative bacteria is in
the permeability of the cell wall
to these "purple colored iodine-dye complexes" when treated with the decolorizing solvent. While Gram-positive bacteria retain purple iodine-dye complexes after the treatment
with the decolorizing agent, Gram-negative bacteria do not retain
complexes when decolorized. To visualize decolorized Gram-negative bacteria, a red counterstain such as safranin is
used after decolorization treatment Appearance of the Gram positive
coccus and Gram negative bacillus at different
stages of the gram staining
procedure are illustrated below:
Preparation of the smear The first consideration
is the correct preparation of the smear.
Make a thin film of the
material on a clean glass slide,
using a sterile loop or swab
for viscous specimens. Air dry, then heat fix the slide by
passing it several times through a flame (the slide should not become too
hot to touch). Failure to follow these directions may cause staining
artifacts and disrupt the normal
morphology of bacteria and cells. To be visible
on a slide, organisms that stain by
the Gram method must be
present in concentrations of a minimum of 104 to 105
organisms/ml of unconcentrated
staining fluid. At lower concentrations, the Gram stain
of a clinical specimen seldom reveals organisms even if the culture
is positive. Smears that are not properly
fixed tend to be washed away
during staining and washing resulting
in the absence of stained
bacteria. In special situations, the following
guidelines may be helpful: When cerebrospinal fluid contains only a few organisms, they are more likely
to be found if a concentrated "thick smear" is examined. To prepare a "thick smear" the specimen is spun
at high speed and a large
drop of sediment (or multiple drops, drying in between
each drop) is placed in
the center of the slide
and allowed to air dry. The
cytocentrifuge may prove
to be useful in concentrating bacteria as well
as in preserving
cell morphology. When fluid specimens such as urine or CSF seem to vanish after the staining procedure, a wax mark, placed near the smeared area
on the slide (same side) after the
staining procedure (to avoid introducing wax artifacts) will reduce frustration
in locating the specimen under the microscope.
The wax mark can be
used for quick focusing. In a grossly bloody specimen,
it may prove difficult to
distinguish microorganisms
from artifacts. After air-drying and heat-fixing this type of
specimen, the added preparatory step of covering it with distilled water, waiting five minutes, and then rinsing,
may cause the red blood
cells to lyse and float off. Staining procedure
Results As shown below, organisms that retain the violet-iodine
complexes after washing in ethanol
stain purple and are termed
Gram-positive, those
that lose this complex stain red from
the safranin counterstain are termed Gram-negative.
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